rK5 induces autophagy in HUVECs. (A) HUVECs cultured in different conditions (5% FBS in M199, 5% FBS in M199 supplemented with 40 ng/mL VEGF, and 10% FBS in M199) were treated with 1.5 μg/mL rK5 for 24 hours and stained with neutral red as described previously.27 Phase-contrast images were recorded at 400× magnification using an Olympus BX60 upright microscope (Olympus, Center Valley, PA). There was an increase in the intensity and size of neutral red–positive vesicles in rK5-treated cells compared with control cultures. (B) Kinetic study of rK5-induced autophagy in endothelial cells. HUVECs were treated with 1.5 μg/mL of rK5 for the indicated time periods (□). Autophagic vesicles were labeled by 0.05 mM MDC and recorded by confocal microscopy at 600× magnification at different time points. The number of autophagic vesicles per cell was determined by an image analysis program as described in “Materials and methods.” Untreated cultures (■) and cultures treated with 1.5 μg/mL purified rAAV-GFP CM (▴). Values are shown as means ± SE. Inset: representative grayscale image of MDC-labeled HUVECs; digital centroid images are shown. Cells were treated by rK5 in the presence of VEGF. Images were recorded after 24 hours of treatment with rK5. (C) TEM analysis of HUVECs treated with 1.5 μg/mL rK5. Treated and control HUVECs were processed for TEM as described previously.20 Images were recorded at 5000× magnification (column 1) and 20 000× magnification (column 2) using a JEOL 1200 EX transmission electron microscope (JEOL, Peabody, MA). Note the presence of double-membrane autophagosomes and single-membrane autolysosomes that contained disintegrated materials clustering at the perinuclear sites (arrows). The nucleus and the cellular membrane structures of both control and treated cell remained intact. (D) rK5-induced autophagy was detected by LC3-GFP. HUVECs were transfected with LC3-GFP using Lipofectamine 2000 (Invitrogen). Transiently transfected cells were treated by 1.5 μg/mL rK5 in the presence of VEGF (40 ng/mL; column 3). Transfected HUVECs cultured with 40 ng/mL VEGF (column 2) or without (column 1) was used as control. After 24 hours of treatment, LC3-GFP–labeled cells (top row) were colocalized with mitochondria labeled with Mitotracker Red (bottom row). Confocal images were obtained at 600× magnification.
